The determination of gene expression patterns in three dimensions with cellular resolution is an important goal in developmental biology. However, the most sensitive, efficient, and widely used staining technique for whole-mount in situ hybridization (WMISH), nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) precipitation by alkaline phosphatase, could not yet be combined with the most precise, high-resolution detection technique, confocal laser-scanning microscopy (CLSM). Here we report the efficient visualization of the NBT/BCIP precipitate using confocal reflection microscopy for WMISH samples of Drosophila, zebrafish, and the marine annelid worm, Platynereis dumerilii. In our simple WMISH protocol for reflection CLSM, NBT/BCIP staining can be combined with fluorescent WMISH, immunostainings, or transgenic green fluorescent protein (GFP) marker lines, allowing double labeling of cell types or of embryological structures of interest. Whole-mount reflection CLSM will thus greatly facilitate large-scale cellular resolution expression profiling in vertebrate and invertebrate model organisms.

Introduction

The most widely used method to determine gene expression patterns in developmental biology is in situ hybridization. Conventional in situ hybridization protocols use digoxigenin (DIG)-11-UTP-labeled RNA probes and detection with an alkaline phosphatase-coupled anti-DIG antibody (1,2,3,4,5). Alkaline phosphatase activity is visualized with the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and color development is enhanced by nitroblue tetrazolium (NBT) yielding an insoluble black-purple precipitate. The sensitivity and precision of alkaline phosphatase-NBT/BCIP staining in whole-mount in situ hybridization (WMISH) is very high, and it often surpasses that of fluorescent staining techniques, especially in the case of weakly expressed genes. However, to date, such samples have been documented only by bright-field microscopy. Being able to image NBT/BCIP samples by confocal laser-scanning microscopy (CLSM) would bring several advantages that greatly facilitate expression profiling in whole embryos, such as enhanced depth of focus and the ability to observe multiple fluorescent signals in isolation or in combination. Here we report a method that allows the detection of conventional WMISH samples by confocal microscopy in zebrafish, Drosophila, and the marine annelid Platynereis dumerilii. The method is based on reflection microscopy that has already been used to detect NBT/BCIP samples and other types of precipitates [e.g., diaminobenzidine (DAB)] in tissue sections and fixed cells (6,7,8,9), but not in whole-mount embryo samples. Our method will greatly facilitate the determination of the extent of gene expression in embryos in three-dimensional (3-D) volume rendering and also allow fluorescent co-staining and colocalization studies.

Materials and Methods

Microscopy

Bright-field images were taken on a Zeiss Axiophot microscope (Carl Zeiss, Jena, Germany) using differential interference contrast (DIC) optics. Confocal images were taken either with a Leica TCS SP2 or a Leica TCS SPE confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a 40× oil-immersion objective with a pinhole of 2 airy units. For whole-mount reflection CLSM, a 633 nm gas laser or a 635 nm diode laser were used, and the detection window was set to 630-640 nm. It was combined either with fluorescent antibody staining or fluorescent tyramide WMISH and confocal detection of fluorescence using appropriate laser lines. Z-projections and 3-D projections of confocal stacks and quantifications of average pixel intensities were done using ImageJ and Imaris 5.5.

Whole-Mount In Situ Hybridization

Platynereis and zebrafish embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) plus 0.1% Tween® 20 (PTW) for 2 h. Drosophila embryos were dechorionated in 50% bleach, fixed in a biphasic mixture of heptane, PBS, and 40% formaldehyde as 1:0.3:0.03 for 20 min and devitellinized in 1:1 heptane, MeOH. Embryos were stored in methanol at -20°C and rehydrated in a 75%/50%/25% methanol/PTW series, 5 min each, rinsed two times for 5 min in PTW and digested for 1 min in 100 µg/mL proteinase K. Embryos were rinsed twice in 2 mg/mL glycine/PTW, post-fixed for 20 min in 4% paraformaldehyde/PTW, and rinsed five times for 5 min in PTW. After prehybridization for 1 h in Hyb-Mix [50% formamide, 5× standard saline citrate (SSC), 50 µg/mL heparin, 0.1% Tween 20, 5 mg/mL torula RNA] at 65°C, DIG-11-UTP-labeled RNA probe, denatured at 80°C for 10 min in Hyb-Mix, was added to the prehybridized embryos. Hybridization was carried out overnight at 65°C. Embryos were washed twice in 50% formamide/2× SSC/0.1% Tween 20, twice in 2× SSC/0.1% Tween 20, and twice in 0.2× SSC/0.1% Tween 20 for 30 min at 65°C each. Embryos were blocked in 5% sheep serum in PTW for 1 h at room temperature and incubated in 1:2000 anti-DIG antibody (Roche, Indianapolis, IN, USA) in PTW/5% sheep serum overnight at 4°C. Optionally, mouse anti-acetyl-ated tubulin (Sigma, St. Louis, MO,USA) and rabbit anti-green fluorescent protein (GFP) antibodies were also added at this step at 1:500. After six PTW washes, 10 min each, embryos were rinsed in staining buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2, 0.1% Tween 20) and stained for several hours in 337.5 µg/mL NBT and 175 µg/mL BCIP in staining buffer. Staining was stopped in pH 7.5 staining buffer, without NBT/BCIP, and embryos were mounted in glycerol. For the immunostained samples, fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) coupled anti-mouse and rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), diluted as 1:250, were used in PTW/5% sheep serum following the NBT/BCIP staining step. Embryos were incubated with secondary antibodies overnight at 4°C, washed in PTW six times for 10 min, and mounted in glycerol with 2.5 mg/mL DABCO.